Serious bacterial infections are a common cause of morbidity and mortality in the U.S. and around the world. The development of relatively antibiotic resistant bacteria has made this problem more difficult to treat with antibiotics.
In the past, antibody therapy has been used successfully to treat bacterial infections. Pneumococcal pneumonia and pneumococcal sepsis responded to treatment with antibody to pneumococcal polysaccharide in the pre-antibiotic era. However, the development of highly effective antibiotics for bacterial infections made this approach obsolete. There were also problems with antibody therapy. Each type of pneumococcal capsule required a specific antiserum, making therapy expensive and cumbersome. In addition, because the antisera were made in horses, toxic immunologic reactions (serum sickness) were a serious side effect. Furthermore, antibody therapy had no effect against other pathogens, such as Staphylococcus or gram-negative bacteria, because of the specificity of the antisera for the pneumoccocal polysaccharide.
The development of antibiotic resistance by bacterial has seriously altered our ability to combat bacterial infections with antibiotics. The development of vancomycin-resistant enterococci, methicillin-resistant Staphylococcus, and multi-drug resistant Gram-negative bacteria has limited the number of effective antibiotics. Though the search for new antibiotics has intensified, additional therapeutic approaches are needed.
Innate immunity is the first line of defense against pathogens. A key component of the mammalian innate immune system is a family of toll like receptors (TLRs). Lipopolysaccharide (LPS), a major component of gram-negative bacteria, activates a variety of cells to produce inflammatory cytokines leading to septic shock in humans. The innate immune mechanism that recognizes LPS involves a transfer of LPS to a pattern recognition molecule CD14 by LBP. Toll like receptor 4 (TLR4) is a type 1 transmembrane protein that has extracellular leucine rich repeats and an intracellular signaling domain that is responsible for LPS signaling. TLR4 is complexed with MD-2, a 22-25 Kd glycoprotein, on the cell surface. A cascade of events leading to maximal cellular activation is likely to involve transferring of LPS by LBP to CD14 and then to TLR4/MD-2. Although CD14 and LBP enhance cellular activation, activation of TLR4 by LPS absolutely requires MD-2.
MD2 is a pattern recognition receptor that binds LPS with a high affinity (an apparent Kd of 65 nM) and without the need for LPS binding protein to catalyze the reaction. It is an extracellular protein that is co-expressed with TLR4, and necessary for TLR4 LPS receptor function. Truncation of MD2 leads to LPS non-responsiveness and a monoclonal antibody that recognizes the MD2/TLR4 complex blocks LPS activation of cells.
MD-2 can be found on the cell surface in association with TLR4 or as a secreted protein. It shares a sequence homology to MD-1, a protein that binds to another TLR family member, RP105, that constitutes an LPS signaling complex on B-cells. MD-2 contributes to ligand recognition of TLR4. It binds LPS with high affinity and discriminates ligand recognition between mouse and human TLR4 to Taxol and lipid IVa. Interaction of cell surface TLR4/MD-2 complex by LPS-induced clustering of TLR4 leads to signal cellular activation.
Although proper glycosylation and trafficking TLR4 to the cell surface requires intracellular association with MD-2, functional TLR4 can be presented on the cell surface without MD-2 in both transfected cells and human airway epithelial cells. These cells can respond to LPS only in the presence of soluble MD-2. While soluble MD-2 (sMD-2) is essential for LPS induced activation of cells expressing only TLR4, high levels of sMD-2 inhibit cellular response to LPS in a whole blood assay or activation of U373 cells, presumably by sequestering LPS. Soluble MD-2 exists as the heterogeneous collection of monomer and oligomers through inter and intra chain disulfide bonds. It has been unclear how the different isoforms function.
Human MD-2 contains 160 amino acids residues, including the N-terminal 17 amino acid signal sequence, with 7 cysteine residues and 2 N-glycosylation sites.